ABSTRACT
Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.
Subject(s)
Achromobacter , Glycine , Glyphosate , Operon , Soil Microbiology , Glycine/analogs & derivatives , Achromobacter/genetics , Operon/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Herbicides , Multigene Family , Kinetics , Gene Expression Regulation, Bacterial/drug effectsABSTRACT
Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3ß-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.
Subject(s)
Cholesterol Oxidase , Phytosterols , Actinobacteria , Cholestanols , Cholesterol Oxidase/chemistry , Dehydroepiandrosterone/chemistry , Hydrogen Peroxide , Pregnenolone , Steroids/chemistryABSTRACT
The genome of an Achromobacter insolitus strain isolated from an agricultural soil polluted with the herbicide glyphosate is reported. The genome size is 6.4 Mb, with an average G+C content of 65.2%. These genomic data could contribute to a better understanding of the biochemistry and regulatory mechanisms of the microbial degradation of glyphosate and aminomethylphosphonate.
